Sureprint g3 human gene expression 8 60k v2 microarray kit

Microarray experiments were performed on parent OVA-BS4 and OVA-BS4 spheroids using the commercially available G4851B human whole GE Microarray kit (SurePrint G3 Human Gene Expression 8 × 60 K v2 Microarray Kit Agilent Technologies) according to manufacturer’s instructions. Fluorescence intensities were measured by Feature Extraction software v11 (Agilent Technologies). Raw data were pre-processed, removing features marked as unreliable by the scanning software in at least 60% of the samples, and afterward normalized using the “quantile” method. Differential analysis was carried out with linear models for microarray analysis [15 (link)], correcting the resulting p-values for multiple testing with the False Discovery Rate (FDR) method [16 ]. Only genes with a corrected p-value of less than 0.01 and regulated at least two fold compared to controls were called significant. In accordance to the MIAME guidelines, raw and processed data have been submitted to the Array Express repository (ID pending).
Gene Enrichment Analysis for selected genes was performed based on a cancer stem cell (CSC)-specific pathway gene list (Additional file 1: Table S1) and a 34 gene-based signature predictive of chemoresistance (Additional file 2: Table S2), using Fisher Exact Test. HUGO gene symbols were used as matching criteria after duplicates removal.

Total RNA was isolated from minced tissue samples using a miRNeasy Mini Kit (217004; Qiagen) as described previously (25 (link)). RNA samples with an RNA integrity number ≥6.0 underwent gene expression profiling (GEP) using a one-color Low Input Quick Amp Labeling Kit (5190-2305; Agilent Technologies) for amplification/fluorescence-labeling and a SurePrint G3 Human Gene Expression 8 × 60K v2 Microarray kit (G4851B; Agilent Technologies) for detecting gene expression. The microarray kit contains 50,599 probes capable of detecting 29,833 genes registered in the Entrez Gene Database of the National Center for Biotechnology Information. Hybridization signals were detected using a DNA Microarray C Scanner (Agilent Technologies), and the scanned images were analyzed using Agilent Feature Extraction v10.7.3.1 software. Microarray analysis was performed in accordance with Minimum Information About a Microarray Experiment guidelines (26 (link)). Data analysis was performed using GeneSpring GX v14.9.1 (Agilent Technologies), the Subio Platform, Excel 2021, and GraphPad Prism v8.3.0. We selected probes to be analyzed according to the reference genome sequence hg19, obtained from the UCSC Genome Browser (27 (link)). Raw signal intensity values were log₂ transformed and normalized to the 75th percentile.